Composition of mannuronic diacid

ABSTRACT

The present invention relates to a mannuronic diacid oligosaccharide composition, comprising a mannuronic diacid of Formula (III) or a pharmaceutically acceptable salt thereof, wherein n is an integer from 1 to 9, m is 0, 1 or 2, and m′ is 0 or 1, and wherein the total weight of mannuronic diacids wherein n=1-5 is 80-95% of the total weight of the composition, and the ratio of the total weight of mannuronic diacids wherein n=1-3 to the total weight of mannuronic diacids wherein n=4-7 is between 1.0 and 3.5.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is Continuation of U.S. application Ser. No.16/474,928, filed on Jun. 28, 2019, which is a U.S. national stagefiling, under 35 U.S.C. § 371(c), of International Application No.PCT/CN2017/118843, filed on Dec. 27, 2017, which claims priority toInternational Application No. PCT/CN2016/113879, filed on Dec. 30, 2016.The entire contents of each of the aforementioned applications areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to an optimal composition of mannuronicdiacids obtained by a biological activity screening method, which usesan animal model of senile dementia to evaluate the effects of differentpolymerization degrees and proportions of mannuronic diacids on thebiological activity thereof. The composition with the best biologicalactivity was finally screened and the desired target substance wasprepared by ultrafiltration membrane separation.

BACKGROUND OF THE INVENTION

Mannuronic diacids have been paid extensive attention due to theirpotential medicinal values. Mannuronic diacids are usually prepared by amulti-step method using alginic acid as a raw material.

The polysaccharide molecule of the raw material, alginic acid, comprisesan M segment formed of D-mannuronic acids linked by β-1,4-glycosidicbonds, a G segment formed of L-guluronic acids linked byα-1,4-glycosidic bonds, and a hybrid MG segment formed of the twosaccharides. The structural formulae of mannuronic acid and guluronicacid are shown in the following Formula (I):

The M and G segments can be separated from the raw material, alginicacid. A common method can be simply described below: alginic acid ispreliminarily degraded to give a polysaccharide mixture ofpolymannuronic acid and polyguluronic acid; the polysaccharide mixtureis subjected to acidic precipitation to remove the polyguluronic acidtherein; and further refinement is conducted to obtain ahomopolymannuronic acid having a purity of 90% or more (hereinafter alsoreferred to as “M-segment intermediate”). See, e.g., the methodsdisclosed in Chinese Patent Application No. 98806637.8 and CN02823707.2.

Oligomannuronic acid can be prepared as follows: the M-segmentintermediate obtained above is subjected to further acidolysis byheating under an acidic condition to obtain a small fragment mannuronicacid polymer having a desired range of molecular weight. In addition,the degradation efficiency can be improved by an oxidative degradationmethod; meanwhile, the reducing end can be oxidized to a ring-openedsaccharic acid, see Chinese Patent Application No. 200580009396.5(Patent literature 1) filed by Meiyu Geng, et al. and U.S. Pat. No.8,835,403 B2 (Patent literature 2). For convenience, Patent literatures1 and 2 are hereinafter collectively referred to as prior patents, whichare incorporated herein by reference in their entirety.

The reaction process of mannuronic diacid disclosed in prior patents canbe represented by the following reaction equation (II), that is, thealdehyde group at position Cl of mannuronic acid at the reducing end ofoligomannuronic acid polysaccharide is oxidized to a carboxyl group.

In the above oxidative conversion process, a commonly used oxidant is analkaline copper sulfate solution, i.e., Fehling's reagent. Prior patentsjust adopt this oxidation method. Specifically, under an alkalinecondition, the reaction substrate polymannuronic acid, i.e., the aboveM-segment intermediate, is added to a copper sulfate solution andreacted in a boiling water bath for 15 minutes to 2 hours. The methoduses Cu²⁺ ions as an oxidizing agent to oxidize the aldehyde group, anda brick-red cuprous oxide precipitate is generated in the reaction. Thisreaction is often used to identify a reduced sugar.

Prior patents disclose that oligomannaric acids have effects againstAlzheimer's disease (AD) and Diabetes Mellitus. The pathogenesis ofAlzheimer's disease and type 2 diabetes is closely related to amyloids(β-amyloid and amylin). Amyloids can aggregate to form proteinoligomers, and can further aggregate to form fibers. These proteinaggregates are cytotoxic, can induce an oxidation reaction in cells todamage mitochondria, and can trigger a cascade reaction such asinflammatory response, causing damage to a large number of neurons andbeta cells, and ultimately leading to onset of Alzheimer's disease andtype 2 diabetes. Oligomannaric acids target amyloids and antagonize thecascade reactions induced by the amyloids, and therefore have theeffects of preventing and treating Alzheimer's disease and type 2diabetes.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a mannuronic diacidoligosaccharide composition, comprising a mannuronic diacid of Formula(III) or a pharmaceutically acceptable salt thereof:

wherein n is an integer from 1 to 9, m is 0, 1 or 2, and m′ is 0 or 1,

and wherein,

the total weight of mannuronic diacids wherein n=1-5 is 80-95% of thetotal weight of the composition, and

the ratio of the total weight of mannuronic diacids wherein n=1-3 to thetotal weight of mannuronic diacids wherein n=4-7 is between 1.0 and 3.5.

Another aspect of the present invention provides a pharmaceuticalcomposition or a health care product comprising the mannuronic diacidoligosaccharide composition of the present invention and, if necessary,a suitable carrier.

A further aspect of the present invention provides a method for treatinga patient with senile dementia, comprising administering an effectiveamount of the mannuronic diacid oligosaccharide composition of thepresent invention to a patient in need thereof.

The mannuronic diacid oligosaccharide composition of the presentinvention is prepared by a method different from that of the prior art.This method of preparation has the advantages of a simple reaction, ahigh content of active ingredient, and no residual reaction reagents. Ithas been experimentally demonstrated that the mannuronic diacidoligosaccharide composition of the present invention can inhibit celldamage, protect nerve cells, and increase cell survival rate. In ananimal model, the mannuronic diacid oligosaccharide composition of thepresent invention can significantly improve the learning and cognitivefunctions of dementia rats. The mannuronic diacid oligosaccharidecomposition of the present invention has potential effects of preventingand treating Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mass spectra of disaccharide, trisaccharide andtetrasaccharide in product A.

FIG. 2 shows mass spectra of pentasaccharide, hexasaccharide andheptasaccharide in product A.

FIG. 3 shows mass spectra of octasaccharide, nonasaccharide anddecasaccharide in product A.

FIG. 4 shows the protective effect of product A at differentconcentrations on AP-induced nerve cell damage.

FIG. 5 shows the protective effect of oligomannaric acid with singlepolymerization degree on Aβ-induced nerve cell damage.

FIG. 6 shows evaluation of the effects of from disaccharide todecasaccharide on an animal model of AD.

FIG. 7 shows effects of the oligosaccharide compositions andhexasaccharide on the number of times AD animals pass through theplatform.

FIG. 8 shows effect of the oligosaccharide compositions andhexasaccharide on swimming distance of AD animals.

FIG. 9 shows the activities of from disaccharide to decasaccharide andcomposition A on a cell co-culture model.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the present invention will be described in detailbelow. However, the present invention is not limited to these specificembodiments. A person skilled in the art can make some modifications andadjustments to the present invention in light of the substantialdisclosure below, and such modifications are also encompassed in thescope of the present invention.

Mannuronic Diacid Oligosaccharide Composition

A first aspect of the present invention relates to a mannuronic diacidoligosaccharide composition, comprising a mannuronic diacid of Formula(III) or a pharmaceutically acceptable salt thereof:

wherein n is an integer from 1 to 9, m is 0, 1 or 2, and m′ is 0 or 1,

and wherein,

the total weight of mannuronic diacids wherein n=1-5 is 80-95% of thetotal weight of the composition, and the ratio of the total weight ofmannuronic diacids wherein n=1-3 to the total weight of mannuronicdiacids wherein n=4-7 is between 1.0 and 3.5.

The mannuronic diacid oligosaccharide composition of the presentinvention is a mixture of mannuronic diacids with differentpolymerization degrees, and the main components thereof are mannuronicdiacid oligosaccharides with a polymerization degree of 2 to 10.According to the prior applications, the most active saccharides inmannuronic diacids are from pentasaccharide to octasaccharide, inparticular hexasaccharide. However, unlike the known prior art, theinventors have found that addition of less active disaccharide totetrasaccharide to the most active pentasaccharide to octasaccharideyields a biological activity better than that of pentasaccharide tooctasaccharide, under the condition of diluting the concentrations ofthe highly active saccharides.

According to a preferred embodiment, in the mannuronic diacidoligosaccharide composition of the present invention, the total weightof mannuronic diacids wherein m+m′=1 or 2 is not less than 50% or more,preferably 60-90%, more preferably 70-90% of the total weight of thecomposition. In particular, in the mannuronic diacid oligosaccharidecomposition of the present invention, the total weight of mannuronicdiacids wherein m+m′=1 is not less than 10%, preferably 30-40% of thetotal weight of the composition. In another preferred embodiment, in themannuronic diacid oligosaccharide composition of the present invention,the total weight of mannuronic diacids wherein m+m′=2 is not less than10%, preferably 30-50% of the total weight of the composition.

According to a preferred embodiment, in the mannuronic diacidoligosaccharide composition of the present invention, the total weightof the mannuronic diacid oligosaccharides wherein n=1-5 is 80-95% of thetotal weight of the composition.

According to a preferred embodiment, in the mannuronic diacidoligosaccharide composition of the present invention, the total weightof the mannuronic diacid oligosaccharides wherein n=1-3 is 20-70% of thetotal weight of the composition.

According to a preferred embodiment, in the mannuronic diacidoligosaccharide composition of the present invention, the ratio of thetotal weight of the mannuronic diacids wherein n=1-3 to the total weightof the mannuronic diacid oligosaccharides wherein n=4-7 is between 1.0and 3.5, preferably between 1.0 and 3.0.

According to a preferred embodiment, in the mannuronic diacidoligosaccharide composition of the present invention, the weightpercentages of mannosonic diacid oligosaccharides with differencepolymerization degrees in the composition are: 5-25% disaccharide,15-30% trisaccharide, 15-25% tetrasaccharide, 10-25% pentasaccharide,5-15% hexasaccharide, 3-10% heptasaccharide, 2-5% octasaccharide, 1-5%nonasaccharide, and 1-5% decasaccharide. In particular, the weightpercentages of the oligosaccharides in the composition are: 10-20%disaccharide, 18-30% trisaccharide, 15-25% tetrasaccharide, 15-20%pentasaccharide, 5-10% hexasaccharide, 3-5% heptasaccharide, 2-3%octasaccharide, 1-3% nonasaccharide, and 1-3% decasaccharide.

In the mannuronic diacid oligosaccharide composition of the presentinvention, the pharmaceutically acceptable salt is a sodium salt or apotassium salt.

Method for Preparing a Mannuronic Diacid Oligosaccharide Composition

The process for preparing mannuronic diacid according to the presentinvention is summarized as follows.

The M-segment intermediate as described above is oxidatively degraded onthe sugar chain in the presence of an oxidizing agent to give oxidizedoligosaccharides with different polymerization degrees. The oxidizedoligosaccharides are characterized in that the mannuronic acids at thereducing end of the oligosaccharides have been oxidized to saccharicacids having 3-6 carbon atoms.

The oxidizing agent which is particularly advantageous to the reactionof the present invention is ozone. During the reaction, the oxidativedegradation reaction of the sugar chain occurs when ozone is introducedinto a solution containing the M-segment intermediate. The temperatureat which the oxidative degradation step is carried out is preferably0-70° C., more preferably 10-45° C. The pH at which the oxidativedegradation step as described above is carried out is 3-13, preferably4-10, more preferably 6-8.

The oxidative degradation reaction using ozone in the present inventionand the oxidative degradation using alkaline copper sulfate (priorpatents) or acid hydrolysis in the presence of hydrogen peroxide andsodium hypochlorite (Chinese Patent Application No. 01107952.5) in theprior art all cause degradation of the sugar chain, but the structuresat reducing ends of sugar chains of the degradation products aredifferent: the oxidative degradation product obtained in the presentinvention, mannuronic diacid, has a diacid structure having 3-6 carbonatoms at the reducing end. Additionally, the process used in theoxidative degradation step of the present invention also offers otheradvantages: 1) the reaction condition is mild, and no special reactioncondition is required; 2) the ozone used can be prepared in situ, andthus the transportation pressure is reduced in industrial production;and 3) after the reaction, the ozone is automatically decomposed intooxygen, and thus there is no harm caused by residual reaction reagentsor environmental pollution. The reaction process is shown in thefollowing equation (IV):

In the schematic diagram of the above reaction equation (IV) and thecompound of Formula (III),

-   -   an oligosaccharide wherein m=2 and m′=1 is a saccharic acid        comprising 6 carbon atoms;    -   an oligosaccharide wherein m=1 and m′=1 or (m=2 and m′=0) is a        saccharic acid comprising 5 carbon atoms;    -   an oligosaccharide wherein m=1 and m′=0 or (m=0 and m′=1) is a        saccharic acid comprising 4 carbon atoms; and    -   an oligosaccharide wherein m=0 and m′=0 is a saccharic acid        comprising 3 carbon atoms.

The above reaction product is desalted by membrane separation to obtainproduct A, as determined by LC-MS structure verification andoligosaccharide proportion measurement. The oligosaccharide proportionis determined by molecular sieve exclusion chromatography in combinationwith multi-angle laser scatterometry. Then, product A is separated bycolumn chromatography to prepare oligosaccharides with singlepolymerization degree: from disaccharide to decasaccharide. Theseoligosaccharides with single polymerization degree are compared forbiological activity in vitro and in vivo. It has been found thathexasaccharide has the best activity among the 9 oligosaccharides, whichis similar to the results of prior patents, e.g., the oligosaccharideactivity results disclosed in prior patent application document 1.

The inventors of the present patent application have found that when theabove 9 oligosaccharides having novel structures are compounded in acertain ratio, a highly active oligosaccharide composition having ahigher activity than the most active hexasaccharide can be obtained. Theproportions of various oligosaccharides in the highly activeoligosaccharide composition need to be combined according to thefollowing proportional relationship:

The total weight of mannuronic diacid oligosaccharides wherein n=1-5 inthe composition is 80-95% of the total weight of the composition, andthe total weight of mannuronic diacid oligosaccharides wherein n=1-3 is20-70% of the total weight of the composition. The ratio of the totalweight of mannuronic diacid oligosaccharides wherein n=1-3 to the totalweight of mannuronic diacid oligosaccharides wherein n=4-7 is between1.0 and 3.5, preferably between 1.0 and 3.0.

The present invention provides a formula for preparing a highly activeoligomannaric acid oligosaccharide composition.

The mannuronic diacid oligosaccharide composition of the presentinvention can inhibit cell damage and protect nerve cells. In an animalmodel, the mannuronic diacid oligosaccharide composition provided by thepresent invention can significantly improve the learning and cognitivefunctions of dementia model animals. Therefore, the mannuronic diacidoligosaccharide composition provided by the present invention haspotential effects of preventing and treating Alzheimer's disease.

In an exemplary embodiment, the method of the present invention includesthe following steps:

(1) Preparation of Mannuronic Diacid Product:

Preparation of M-segment intermediate. As described above, the startingmaterial M-segment intermediate used in the present invention can beproduced by a method known in the prior art, e.g., the methods disclosedin Chinese Patent Application No. 98806637.8 and CN02823707.2. A commonmethod can be simply described below: alginic acid is preliminarilydegraded to give a polysaccharide mixture of polymannuronic acid andpolyguluronic acid; the polysaccharide mixture is subjected to acidicprecipitation to remove the polyguluronic acid therein; and furtherrefinement is conducted to obtain a homopolymannuronic acid having apurity of 90% or more, i.e., an M-segment intermediate.

Ozone oxidative degradation. The M-segment intermediate is dissolved inan appropriate amount of water and stirred at room temperature or underheating. Ozone is continuously charged to initiate the reaction. The pHof the reaction can be adjusted to 3-13, preferably 4-10, morepreferably 6-8 by dropwise adding dilute hydrochloric acid or a diluteNaOH solution. The temperature is preferably 0-70° C., more preferably10-45° C. After the reaction is completed, the charging of ozone isstopped and the pH is adjusted to neutral.

Membrane separation and purification. The reaction product obtainedabove is formulated into a solution at a concentration of about 10%, andseparated by a molecular cut-off membrane to remove degradation productsbelow monosaccharide, and collect the retentate. The molecular cut-offmembrane used has an MWCO of 1000-3000 Da, preferably 2000 Da. Thecollected liquid is concentrated on a rotary evaporator and dried undervacuum to obtain an oligomannuronic diacid mixture. These products arefound to be compositions comprising oligosaccharides, i.e., fromdisaccharide to decasaccharide, with contents being within certainranges. Three compositions, A, B and C, were prepared according to theforegoing method. The proportions and structures of oligosaccharides inthese compositions were confirmed in Examples 1-3.

(2) Preparation of Oligosaccharides with a Single Polymerization Degree

The oligosaccharide mixture obtained in step (1) is dissolved to aconcentration of about 10%, separated on a P6 gel chromatographiccolumn, and subjected to ultraviolet detection to collect each effluentcomponent. The components having the same polymerization degree arecombined. Nine components of from disaccharide to decasaccharide arecollected, desalted by G10 gel column chromatography, concentrated on arotary evaporator, and dried under vacuum. The specific purification andpreparation processes are shown in Example 4. These operations of columnchromatography, desalting and drying are known to those skilled in theart.

The 9 oligosaccharides with single polymerization degree were evaluatedfor pharmacological activity in an animal model of senile dementia. Itwas found that hexasaccharide had the best activity. See Example 4 fordetails.

(3) Comparison of Activities of Oligosaccharide Compositions

The oligosaccharides with single polymerization degree as prepared inthe above step (2) are compounded in the mass percentages as shown inthe following table to obtain a fourth composition, i.e., composition D.The proportions of oligosaccharides in the three oligosaccharidecompositions A, B and C from the above step (1) and composition D areshown in the following table:

tri- tetra- penta- hexa- hepta- octa- nona- deca- disaccharidesaccharide saccharide saccharide saccharide saccharide saccharidesaccharide saccharide A 19% 25% 22% 13%  9% 6% 3% 2% 1% B 24% 25% 19%12%  9% 5% 3% 2% 1% C  8% 20% 28% 19% 13% 6% 3% 2% 1% D  5% 30% 20% 20% 5% 5% 5% 5% 5%

The above four compositions and the hexasaccharide purified in step (2)are compared for pharmacological activities. The results show that thefour oligosaccharide compositions A, B, C and D are significantly moreactive than hexasaccharide that has the best activity in theoligosaccharides with single polymerization degree. It can be seen thata single oligosaccharide can play a synergistic effect aftercompounding. After compounding, the oligosaccharides that are lessactive, such as disaccharide and trisaccharide, are more active thanhexasaccharide.

In summary, the present invention provides a method for preparing ahighly active mannuronic diacid oligosaccharide composition, comprisingan oxidative degradation reaction using the M-segment intermediate as araw material in the presence of ozone, and separation and purificationof the reaction product through ultrafiltration membrane. Thepreparation process involves a simple production process and a highyield, and the reaction product can be easily purified to obtain aproduct having a good activity. The inventors also reveal ranges of themass percentages and proportions of various oligosaccharides in thehighly active composition. The significance of the preparation processprovided by the present invention lies in that a mannuronic diacidhaving a novel structure, i.e., a diacid residue having 6 possiblestructures at the reducing end of the sugar chain, is obtained, and thatthe prepared oligosaccharide composition comprises moderate proportionsof various oligosaccharides and has a strong biological activity.

The present invention further provides a medicament or health careproduct comprising an mannuronic diacid oligosaccharide composition asdescribed above, and optionally a pharmaceutically acceptable carrier orexcipient.

Methods for preparing oligosaccharide combination drugs containingactive ingredients in various proportions are known, or apparent tothose skilled in the art from the disclosure of the present invention,for example, as described in Remington's Pharmaceutical Sciences,Martin, E. W., ed., Mack Publishing Company, 19th ed. (1995). Methodsfor preparing the pharmaceutical composition comprise incorporation ofsuitable pharmaceutical excipients, carriers, diluents and the like.

The pharmaceutical preparation of the present invention is prepared by aknown method, including conventional mixing, dissolving or lyophilizing.

The pharmaceutical composition of the present invention can beadministered to a patient via a variety of routes suitable for thechosen mode of administration, such as orally or parenterally (viaintravenous, intramuscular, topical or subcutaneous routes).

Accordingly, the combination drug of the present invention can beadministered systemically, for example, orally, in combination with apharmaceutically acceptable carrier such as an inert diluent or anedible carrier. It may be enclosed in hard or soft shell gelatincapsules, or it may be compressed into tablets. For oral therapeuticadministration, the active compound of the present invention may beincorporated with one or more excipients and used in the form ofswallowable tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Theproportion of the compositions and preparations may, of course, bevaried and may be in a range of from about 1% to about 99% by weight ofa given unit dosage form. The amount of an active compound in suchtherapeutically useful compositions is such that an effective dosagelevel can be obtained.

The tablets, troches, pills, capsules and the like may also contain: abinder such as gum tragacanth, acacia, corn starch or gelatin; anexcipient such as dicalcium phosphate; a disintegrating agent such ascorn starch, potato starch, alginic acid and the like; a lubricant suchas magnesium stearate; and a sweetening agent such as sucrose, fructose,lactose or aspartame; or a flavoring agent such as peppermint, oil ofwintergreen, or cherry flavoring. When the unit dosage form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier such as vegetable oil or polyethylene glycol. Variousother materials may be presented as coatings or to otherwise modify thephysical form of the solid unit dosage unit. For instance, tablets,pills, or capsules may be coated with gelatin, wax, shellac, or sugar.Syrups or elixirs may contain the active compound, sucrose or fructoseas a sweetening agent, a methylparaben or propylparaben as apreservative, a dye and flavoring agent such as cherry or orange flavor.Of course, any material used for preparing any unit dosage form shouldbe pharmaceutically acceptable and non-toxic in the amounts employed. Inaddition, the active compound may be incorporated into sustained-releaseformulations and sustained-release devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or a salt thereof can be prepared in water optionally mixedwith a non-toxic surfactant. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, triacetin, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powders ofthe active ingredient (optionally encapsulated in liposomes) included inan extemporaneous preparation of a sterile solution or dispersionsuitable for injection or infusion. In all cases, the final dosage formmust be sterile, liquid, and stable under the conditions of manufactureand storage. The liquid carrier can be a solvent or a liquid dispersionmedium comprising, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, liquid polyethylene glycol, and the like), vegetableoils, non-toxic glyceride, and suitable mixtures thereof. The properfluidity can be maintained, for example, by formation of liposomes, bythe maintenance of the required particle size in the case of dispersion,or by the use of surfactants. The action of anti-microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. In the case of sterile powders for the preparation ofsterile injectable solution, the preferred methods of preparation arevacuum drying and the freeze-drying technique which yield a powder ofthe active ingredient plus any additional desired ingredient frompreviously sterile-filtered solution thereof.

Useful solid carriers include pulverized solids (e.g., talc, clay,microcrystalline cellulose, silica, alumina, etc.). Useful liquidcarriers include water, ethanol, ethylene glycol or awater-ethanol/ethylene glycol mixture. The combination drug of thepresent invention may be dissolved or dispersed in the carrier in aneffective amount, optionally with the aid of a non-toxic surfactant.Adjuvants (such as fragrances) and additional antimicrobial agents canbe added to optimize the properties for a given use.

Thickeners (such as synthetic polymers, fatty acids, fatty acid saltsand esters, fatty alcohols, modified celluloses or modified inorganicmaterials) can also be used with liquid carriers to form coatablepastes, gels, ointments, soap, etc., which can be directly applied tothe user's skin.

The therapeutically required amount of the compound or a mixture thereofdepends not only on the compound per se, but also on the mode ofadministration, the nature of the disease to be treated, and the age andcondition of the patient, ultimately depending on the decision of theattending physician or clinician.

The above preparations may be present in unit dosage form, which is aphysically discrete unit containing a unit dose, and is suitable foradministration to human and other mammalian bodies. The unit dosage formcan be a capsule or tablet, or a plurality of capsules or tablets. Theamount of unit dose of the active ingredient may vary or be adjustedbetween about 0.1 and about 1000 mg or more, depending on the particulartreatment involved.

Animal Model and Steps for Evaluating Efficacy and Activity

1. Animal model for evaluating efficacy against AD: An AD model isinduced by unilateral intraventricular injection of Aβ, and learning andmemory behaviors of the AD model rats are evaluated by the Morris watermaze test.

Male Wistar rats are used, each weighing between 180 and 220 g.Randomization: a sham-operation control group, a model group, and dosinggroups, 14 animals per group. The rats are anesthetized byintraperitoneal injection of pentobarbital sodium (40 mg/kg) and fixedon a stereotaxic apparatus. The skin is routinely prepared, sterilized,cut, and the anterior fontanel is exposed. The hippocampal CA1 region islocated at a position “3.0 mm after the anterior fontanel, 2.2 mm nextto the raphe, and 2.8 mm under the dura mater” as described in theStereotaxic Map of Rat Brain, Xinming Bao and Siyun Shu, Beijing,People's Medical Publishing House, 1991, 28. For the model group and thedosing groups, 5 μl of aggregated Aβ (A131-40 is formulated in a PBSsolution to 1.4 mg/mL, and incubated in an incubator at 37° C. for 5days to form an aggregated state) is slowly injected into the righthippocampal CA1 region with a micro-injector needle vertical to theskull, in a flow rate of 1 μL/min. After the injection is completed, theneedle is left for 5 min, such that Aβ can be sufficiently dispersed.Then, the needle is slowly withdrawn. The surgical incision is suturedand kept warm for recovery. The control group receives the sameprocedure except that an equal amount of sterile PBS is injected. Thecorresponding drug is administered 7 days prior to the operation, andthe administration is continued until the end of the experiment.

The Morris water maze test is performed on day 11 after the operation.

Place navigation test: Each group of rats is trained once a day for 5consecutive days, i.e., receives a place navigation test. The time takenby the animals to find the platform (i.e., escape latency) is recorded.The rats that fail to find the platform in about 90 s are guided to swimto the platform in a straight line direction and stand on the platformfor 30 s, to induce their learning and memory.

Spatial probe test: On the second day after the end of the placenavigation test, the platform is removed, and the rats are placed intowater from the place of entry. The number of times the animals passthrough the platform and the percentage of the swimming distance in thequadrant where the platform is located relative to the total distanceare recorded. The learning and memory functions of the animals areevaluated.

2. Model for evaluating cell viability: SH-SY5Y cells (neuroblastomacells) are seeded in a 96-well plate (3000 cells/well). After 24 hr, themedium is removed and a drug is added for pretreatment for 0.5 hr(formulated in a serum-free culture medium; 3 replicates per dose).Then, aggregated Aβ1-42 (Aβ1-42 is formulated in a PBS solution to 1mg/mL, and incubated in an incubator at 4° C. for 24 hr to form anaggregated state, at a final concentration of 2 μM) is added andincubated for 48 hr. The cell viability is detected by CCK8.

Advantages of the present invention are further illustrated in thefollowing non-limiting examples. However, the specific materials andamounts thereof as well as other experimental conditions used in theexamples should not be construed as limiting the present invention. Theparts, proportions, percentages, and the like in the present inventionare all expressed by mass unless otherwise specified.

EXAMPLES Example 1

Step 1): Preparation of a Mannuronic Diacid Oligosaccharide Mixture

An M-segment intermediate was prepared by the method disclosed in priorpatents. The specific operations are simply described below: 5 Kg ofsodium alginate was formulated into a ˜10% solution, and the pH wasadjusted to about 3.0 by adding dilute hydrochloric acid. The solutionwas heated to 80° C., and stirred. It was allowed to react for 10 hrbefore the heating was stopped. After cooling to room temperature, thepH was adjusted to 9.0 by adding NaOH, and further adjusted to 2.85 byadding dilute hydrochloric acid. The solution was centrifuged at 5000rpm for 10 min. The supernatant was collected, and adjusted to pH 1.0 byadding HCl. After centrifugation, the precipitate was collected,concentrated on a rotary evaporator, and dry in vacuo to give 1500 g ofthe M-segment intermediate. 500 g of the M-segment intermediate wasweighed, and dissolved in distilled water to prepare a solution in avolume of 5 L. The solution was adjusted to pH 6.5 with NaOH, and heatedin a water bath to control the reaction temperature at 75° C. The gasflow rate at the outlet of an oxygen cylinder and the power of an ozonegenerator were adjusted such that ozone was fed into the reactionsolution at a mass concentration flow rate of 8 g/hr. After 4 hr ofreaction, the feeding of ozone was stopped, and a suitable amount ofwater was added to adjust the concentration of the solution to about10%. The solution was filtered through an ultrafiltration membrane witha molecular weight cut-off of 2,000 Da to collect a retentate. Thecollected liquid was concentrated on a rotary evaporator and dried undervacuum to obtain 350 g of mannuronic diacid product A.

Step 2): Analysis of Proportions and Structures of Oligosaccharides withVarious Polymerization Degrees in Mannuronic Diacid Product A

100 mg of the above dried mannuronic diacid product A was accuratelyweighed, dissolved in water to a concentration of 10 mg/mL, and passedthrough a 0.22 um filter membrane to obtain a test sample solution. Theproportions of oligosaccharides with different polymerization degrees inthe composition were determined by Superdex peptide molecular exclusionchromatography (GE Co.) in combination with multi-angle laser lightscattering (MALS, Wyatt Co.). The experimental conditions were asfollows:

-   -   Chromatographic column: Superdex peptide 10/300 Gl    -   Mobile phase: 0.1 mol/L NaCl    -   Injection volume: 10 μL    -   Flow rate: 0.3 mL/min

Test results: from disaccharide to decasaccharide were represented bydp2-dp10, respectively. dp2 was 19%, dp3 was 25%, dp4 was 22%, dp5 was13%, dp6 was 9%, dp7 was 6%, dp8 was 3%, dp9 was 2%, and dp10 was 1%.

Step 3): LC-MS Analysis of Structures of Oligosaccharides with VariousPolymerization Degrees in Mannuronic Diacid Product A

Experimental Conditions:

-   -   Chromatographic column: Superdex peptide 10/300 Gl    -   Mobile phase: 20% methanol+80% 80 mmol/L NH₄Ac    -   Flow rate: 0.1 mL/min    -   Column temperature: 25±0.8° C.    -   Mass spectrometry conditions: Agilent 6540 QTOF; ion source: ESI        collision voltage 120 V; negative ion mode. The width of        acquired signal (m/z) was 100-1000.

The mass spectra of oligosaccharides with various polymerization degreesare shown in FIGS. 1-3. Various signal peaks in the mass spectra wereassigned, confirming the molecular structures of all oligosaccharides inproduct A, i.e., the structure as shown in Formula (III). The signalassignments and the structures corresponding to the signals are shown inTable 1 below.

TABLE 1 6 diacid structures of oligosaccharides with differentpolymerization degrees in product A and their mass-to-charge ratios inmass spectra Mass-to-charge ratio (m/z) Molecular Molecular n = 1 n = 2n = 3 n = 4 n = 5 No. structure formula [M − l]⁻ [M − l]⁻ [M − l]⁻ [M −l]⁻ [M − l]⁻ 1

(C₆H₈O₆)_(n)C₆H₁₀O₈ N = 1-9 385 561 737 913 1089 2

(C₆H₈O₆)_(n)C₅H₈O₇ N = 1-9 355 531 707 883 1059 3

(C₆H₈O₆)_(n)C₅H₈O₇ N = 1-9 355 531 707 883 1059 4

(C₆H₈O₆)_(n)C₄H₆O₆ N = 1-9 325 501 677 853 1029 5

(C₆H₈O₆)_(n)C₄H₆O₆ N = 1-9 325 501 677 853 1029 6

(C₆H₈O₆)_(n)C₃H₄O₅ N = 1-9 295 471 647 823 999 Mass-to-charge ratio(m/z) n = 6 n = 7 n = 8 n = 9 No. [M − l]⁻ [M − 2]²⁻ [M − 2]²⁻ [M − 2]²⁻1 1265 720 808 896 2 1235 705 793 881 3 1235 705 793 881 4 1205 690 778866 5 1205 690 778 866 6 1175 675 763 851

It was found from the above mass spectrometric structural analysis thatthe mannuronic acid at the reducing end of the sugar chain in product Awas oxidized to a saccharic acid structure (see Formula III), whichcould be a mannaric acid structure comprising 6 carbon atoms (m+m′=3),with a content of about 10-30%, or a decarboxylation product of mannaricacid, i.e., a saccharic acid comprising 5 carbon atoms (m+m′=2) (30-50%)and a saccharic acid comprising 4 carbon atoms (m+m′=1) (30-40%).

Step 4) Evaluation of Pharmacological Activity

1. Protective Effect of Product A on Aβ-Induced Nerve Cell Injury

The test was conducted according to the “model for evaluating cellviability”, and the experimental procedure was as follows: SH-SY5Y cells(neuroblastoma cells) were seeded in a 96-well plate (3000 cells/well).After 24 hr, the medium was removed, and for the dosing groups, 10 μLper well of a drug (10 mg/mL) was added for pretreatment for 0.5 hr(formulated in a serum-free culture medium; 3 replicates per dose).Then, aggregated Aβ 1-42 (Aβ1-42 was formulated in a PBS solution to 1mg/ml, and incubated in an incubator at 4° C. for 24 hr to form anaggregated state, at a final concentration of 2 μM) was added andincubated for 48 hr. The cell viability was detected by CCK8.

The results showed that treatment of SH-SY5Y cells with 2 μM Aβ1-42could induce significant cell damage and decreased cell viability after48 hours, while 25, 50 and 100 m/mL product A could significantlyinhibit Aβ-induced decrease in cell viability; see FIG. 4. The aboveresults indicate that product A can protect nerve cells from the toxiceffects of Aβ at a low concentration (25 μg/mL), a medium concentration(50 m/mL), and a high concentration (100 m/mL).

Example 2

100 g of the M-segment intermediate from Example 1 was weighed, anddissolved in distilled water to prepare a solution in a volume of 0.8 L.The solution was adjusted to pH 4.0 with NaOH, and the reaction wascarried out at room temperature (25° C.). The gas flow rate at theoutlet of an oxygen cylinder and the power of an ozone generator wereadjusted such that ozone was fed into the reaction solution at a massconcentration flow rate of 1 g/hr. After 10 hr of reaction, the feedingof ozone was stopped, and a suitable amount of water was added to adjustthe concentration of the solution to about 15%. The solution wasfiltered through an ultrafiltration membrane with a molecular weightcut-off of 1,000 Da to collect a retentate. The collected liquid wasconcentrated on a rotary evaporator and dried under vacuum to obtain 80g of mannuronic diacid product B.

The proportions of oligosaccharides with various polymerization degreesin B were determined by Superdex peptide molecular exclusionchromatography (GE Co.) in combination with multi-angle laser lightscattering (MALS, Wyatt Co.). The measurement method was the same asthat in Example 1. Test results: from disaccharide to decasaccharidewere represented by dp2-dp10, respectively. dp2 was 24%, dp3 was 25%,dp4 was 19%, dp5 was 12%, dp6 was 9%, dp7 was 5%, dp8 was 3%, dp9 was2%, and dp10 was 1%.

Example 3

100 g of the M-segment intermediate of Example 1 was weighed, anddissolved in distilled water to prepare a solution in a volume of 1.5 L.The solution was adjusted to pH 9.0 with NaOH, and the reaction wascarried out in a water bath at 45° C. The gas flow rate at the outlet ofan oxygen cylinder and the power of an ozone generator were adjustedsuch that ozone was fed into the reaction solution at a massconcentration flow rate of 3 g/hr. After 2 hr of reaction, the feedingof ozone was stopped, and a suitable amount of water was added to adjustthe concentration of the solution to about 5%. The solution was filteredthrough an ultrafiltration membrane with a molecular weight cut-off of3,000 Da to collect a retentate. The collected liquid was concentratedon a rotary evaporator and dried under vacuum to obtain 60 g ofmannuronic diacid product C.

The proportions of oligosaccharides with various polymerization degreesin C were determined by Superdex peptide molecular exclusionchromatography (GE Co.) in combination with multi-angle laser lightscattering (MALS, Wyatt Co.). The measurement method was the same asthat in Example 1. Test results: from disaccharide to decasaccharidewere represented by dp2-dp10, respectively. dp2 was 8%, dp3 was 20%, dp4was 28%, dp5 was 19%, dp6 was 13%, dp7 was 6%, dp8 was 3%, dp9 was 2%,and dp10 was 1%.

Example 4

Step 1) Preparation of Mannuronic Diacid Oligosaccharide with SinglePolymerization Degree, which was as Follows:

1. Sample Preparation: 300 g of mannuronic diacid product A prepared inExample 1 was dissolved in water to prepare 1000 mL of a concentratedsolution, which was placed in a refrigerator at 4° C. for use. For eachuse, 50 mL of the solution was 1:2 diluted with water, and then suctionfiltered through a 0.22 um ultrafiltration membrane.

2. Chromatographic separation conditions: The chromatograph was AKTApure 150 (purchased from GE Co.) equipped with a UV detector and anautomatic collector. Separation chromatographic column: 1.2 kg of BioGelP6 (purchased from Bio-Rad Co.) was mixed with deionized water, vacuumdegassed, manually filled into a glass column (inner diameter: 10 cm),rinsed with 10 column volumes of pure water. The chromatographic columnbed was stable and the height was 1.0 m. Then, the mobile phase waschanged to a 0.02 M NaCl solution, and after equilibration with 10column volumes, sample loading was initiated.

3. Loading and Separation: The flow rate of the pump was set at 1mL/min. After 100 mL of the sample solution was pumped to the top of thecolumn through the chromatograph's own pump, it was switched to themobile phase and eluted at a flow rate of 5 mL/min. After outflow of thedead water volume, automatic collection was initiated and 50 mL wascollected per tube.

4. The sample loading was repeated, and after 20 repetitions ofpreparation, the same fractions were combined, concentrated on a rotaryevaporator, and lyophilized to obtain a total of 9 oligosaccharides withsingle polymerization degree from disaccharide to decasaccharide.

Step 2) Evaluation of Pharmacological Activity

The Pharmacological Activities of Oligomannaric Acid Oligosaccharideswith Single Polymerization Degree were Evaluated as Follows:

1. Protective Effects of Oligosaccharides on Aβ-Induced Nerve CellInjury

The experiment was carried out in the same manner as described inExample 1, and the oligosaccharide solutions were prepared at aconcentration of 10 mg/mL.

The results showed that treatment of SH-SY5Y cells with 2 μM Aβ1-42could induce significant cell damage and decreased cell viability after48 hours, while all the mannuronic diacid oligosaccharides with singlepolymerization degree had a tendency to inhibit Aβ-induced cell damage.The mannuronic diacid oligosaccharides with a polymerization degree of4-10 (the final concentration of the drugs was 25 μg/mL) couldsignificantly protect nerve cells from the toxic effects of AP, in whichthe oligosaccharides with four polymerization degrees of 5-8 had bettereffects, and hexasaccharide had the best activity; see FIG. 5.

2. Effects of Oligosaccharides on the Learning and Memory ImpairmentModel Induced by Right Intraventricular Injection of Aβ1-40 in Rats

The experimental procedure was carried out on 10 g of each ofdisaccharide to decasaccharide according to the method for “animal modelfor evaluating efficacy against AD”.

Due to the large number of oligosaccharic acids with singlepolymerization degree, the experiment was completed in multiple batches.The comparison and evaluation of the efficacies of variousoligosaccharides was conducted by calculating the percentage of thenumber of times the animals in each group passed through the platformrelative to the number of times the sham-operation control animalspassed through the platform. The results showed that the number ofpassages through the platform was significantly reduced in the modelgroup as compared to the sham-operation control group. Eacholigosaccharide with single polymerization degree had a tendency toincrease the number of passages through the platform. The mannuronicdiacid oligosaccharides with single polymerization degree of 4-10 couldsignificantly increase the number of passages through the platform, inwhich the oligosaccharides with four polymerization degrees of 5-8 hadbetter effects, and hexasaccharide had the best activity; see FIG. 6.

Example 5

A pharmacological activity evaluation was conducted between thecompositions and hexasaccharide to examine the synergistic effect of theoligosaccharides with different polymerization degrees in thecompositions and the range of proportions of the oligosaccharides.

Sample Preparation: The mannuronic diacid oligosaccharides with singlepolymerization degree as prepared in Example 4 were accurately weighedfrom disaccharide to decasaccharide by the polymerization degree. Theweight of each saccharide used was as follows: 0.5 g of disaccharide,3.0 g of trisaccharide, 2.0 g of tetrasaccharide, 2.0 g ofpentasaccharide, 0.5 g of hexasaccharide, 0.5 g of heptasaccharide, 0.5g of octasaccharide, 0.5 g of nonasaccharide, and 0.5 g ofdecasaccharide. They were mixed to obtain 10 g of composition product D.

The proportions of oligosaccharides in products A, B, and C prepared inExamples 1, 2, and 3, respectively, and product D prepared in thepresent Example are shown in Table 2 below.

TABLE 2 Percentages of oligosaccharides in the mannuronic diacidoligosaccharide composition products proportion com- di- trisa- tetra-penta- hexa- hepta- octa- nona- deca- bination saccharide ccharidesaccharide saccharide saccharide saccharide saccharide saccharidesaccharide A 19% 25% 22% 13%  9% 6% 3% 2% 1% B 24% 25% 19% 12%  9% 5% 3%2% 1% C  8% 20% 28% 19% 13% 6% 3% 2% 1% D  5% 30% 20% 20%  5% 5% 5% 5%5%

10 g of each of the above samples A, B, C, and D was used to compare thepharmacological activities of these compositions and hexasaccharide (6T) according to the method described in “animal model for evaluatingefficacy against AD”.

In the experiment, as compared to the sham-operation control group, theanimals in the model group had significantly prolongedplatform-searching latency, indicating that the evaluation modeling wassuccessful. As compared to the model group, each dosing group hadsignificantly shortened platform-searching latency.

There was one resting day after the end of the place navigationtraining. Then, the platform was removed and a spatial probe test wascarried out to observe the number of times animals passed through theplatform and the percentage of the swimming distance in the quadrantwhere the platform was originally located relative to the totaldistance, and evaluate the memory function of the animals. The resultsshowed that the number of passages through the platform wassignificantly reduced in the model group and significantly increased inthe dosing groups as compared to the sham-operation control group, asshown in FIG. 7. The percentage of the swimming distance in the quadrantwhere the platform was originally located relative to the total distanceshowed a similar tendency to the number of passages through theplatform. As compared to the sham-operation control group, thepercentage of the swimming distance in the quadrant where the platformwas originally located relative to the total distance was significantlyreduced in the model group, and was significantly increased in thedosing groups, as shown in FIG. 8.

The experimental results showed that the respective pharmacologicalactivities of oligosaccharide compositions A, B, C and D were still verystrong on day 4, and stronger than the activity of hexasaccharide with asingle polymerization degree, suggesting a synergy between theoligosaccharides in the compositions.

Example 6

A cell co-culturing technique was used to further evaluate theactivities of various oligosaccharides with single polymerization degreeand the compositions.

Suitable amounts of the oligosaccharides with single polymerizationdegree as prepared in Example 4 and the oligosaccharide compositionproduct A prepared in Example 1 were accurately weighed, and dissolvedin PBS to prepare test drug solutions at a concentration of 10 mg/mL.

The cell co-culturing experiment was substantially the same as the cellculturing method in foregoing Example 1 and Example 4. The maindifference lies in that the cell co-culturing technique mimics theinteraction of different cells in vivo. Considering that in vivo cellsmight interact with each other through a signaling pathway, in order tobe closer to the in vivo environment, and simulate the interactionbetween different cells during development of AD, microglial cells wereintroduced during the culture. The specific experimental procedure wasas follows: SH-SY5Y cells (neuroblastoma cells) were seeded in a 24-wellplate (12,000 cells/well), and BV-2 cells (microglial cells) were seededinto the upper chamber at a concentration of 15,000 cells/well. After 24hr, the medium was removed, and the test drug solutions were added tothe lower chamber to obtain a final drug concentration of 25 μg/mL.After 0.5 hr of treatment (formulated in a serum-free culture medium; 3replicates per drug solution), aggregated A131-42 (A01-42 was formulatedin a PBS solution to 1 mg/mL, and incubated in an incubator at 4° C. for24 hr to form an aggregated state, at a final concentration of 2 μM) wasadded and incubated for 48 hr. The viability of SH-SY5Y cells in thelower chamber was detected by CCK8.

After 48 hours, the model group was compared with the normal controlgroup. The former exhibited significant damage and reduced cell survivalrate. The dosing groups showed the effect of inhibiting Aβ-induced celldamage. In particular, the activity of product A was significantlybetter than the activities of other 9 oligosaccharides with singlepolymerization degree, as shown in FIG. 9. The co-cultured cell modelcan identify the difference in activity between the composition and theoligosaccharides with single polymerization degree, possibly because asynergistic effect can occur between cytokines released from themicroglial cells and the oligosaccharides with different polymerizationdegrees in the composition, thereby increasing the activity of theoligosaccharide composition.

1. A mannuronic diacid saccharide composition, comprising a mixture ofmannuronic diacid of Formula (III) or a pharmaceutically acceptable saltthereof:

wherein n is an integer from 1 to 9, m is 0, 1 or 2, and m′ is 0 or 1,and wherein, the total weight of mannuronic diacids wherein n=1-5 is80-95% of the total weight of the composition; and the ratio of thetotal weight of mannuronic diacids wherein n=1-3 to the total weight ofmannuronic diacids wherein n=4-7 is between 1.0 and 3.5; and the totalweight of mannuronic diacids wherein m+m′=1 and 2 is equal to or greaterthan 50% of the total weight of the composition.
 2. The mannuronicdiacid saccharide composition according to claim 1, wherein the totalweight of mannuronic diacids wherein m+m′=1 and 2 is 60-90% of the totalweight of the composition.
 3. The mannuronic diacid saccharidecomposition according to claim 1, wherein the total weight of mannuronicdiacids wherein m+m′=1 is not less than 10% of the total weight of thecomposition.
 4. The mannuronic diacid saccharide composition accordingto claim 1, wherein the total weight of mannuronic diacids whereinm+m′=2 is not less than 10% of the total weight of the composition. 5.The mannuronic diacid saccharide composition according to claim 1,wherein the total weight of the mannuronic diacids wherein n=1-5 is80-95% of the total weight of the composition.
 6. The mannuronic diacidsaccharide composition according to claim 1, wherein the total weight ofthe mannuronic diacids wherein n=1-3 is 20-70% of the total weight ofthe composition.
 7. The mannuronic diacid saccharide compositionaccording to claim 1, wherein the ratio of the total weight of themannuronic diacids wherein n=1-3 to the total weight of the mannuronicdiacids wherein n=4-7 is between 1.0 and 3.5.
 8. The mannuronic diacidsaccharide composition according to claim 7, wherein the ratio of thetotal weight of the mannuronic diacids wherein n=1-3 to the total weightof the mannuronic diacids wherein n=4-7 is between 1.0 and 3.0.
 9. Themannuronic diacid saccharide composition of claim 1, wherein the weightpercentages of mannosonic diacids with difference polymerization degreesin the composition are: 5-25% n=1, 15-30% n=2, 15-25% n=3, 10-25% n=4,5-15% n=5, 3-10% n=6, 2-5% n=7, 1-5% n=8, and 1-5% n=9.
 10. Themannuronic diacid saccharide composition according to claim 9, whereinthe weight percentages of mannosonic diacids with differencepolymerization degrees in the composition are: 10-20% n=1, 18-30% n=2,15-25% n=3, 15-20% n=4, 5-10% n=5, 3-5% n=6, 2-3% n=7, 1-3% n=8, and1-3% n=9.
 11. The mannuronic diacid saccharide composition of claim 1,wherein the pharmaceutically acceptable salt is a sodium salt or apotassium salt.
 12. A pharmaceutical composition or health care product,comprising an effective amount of the mannuronic diacid saccharidecomposition claim 1, and, optionally, a suitable carrier. 13-14.(canceled)
 15. A method for treating a patient with senile dementia,comprising administering an effective amount of the mannuronic diacidsaccharide composition of claim 1 to a patient in need thereof.
 16. Amethod for preparing a mannuronic diacid saccharide and a compositionthereof by ozone oxidative degradation, by which the components orcomposition as defined in claim 1 is prepared.
 17. The method forpreparing an oligosaccharide and a composition thereof by ozoneoxidative degradation according to claim 16, wherein: the oxidationreaction is carried out at a temperature of 0-70° C.; and the oxidativedegradation step is carried out at a pH of 3-13.
 18. The method forpreparing a mannuronic diacid saccharide composition according to claim16, wherein: the oxidation reaction is carried out at a temperature of10-45° C.; and the oxidative degradation step is carried out at a pH of4-10.
 19. The mannuronic diacid saccharide composition according toclaim 1, wherein the total weight of mannuronic diacids wherein m+m′=1and 2 is 70-90% of the total weight of the composition.
 20. Themannuronic diacid saccharide composition according to claim 1, whereinthe total weight of mannuronic diacids wherein m+m′=1 is 30-40% of thetotal weight of the composition.
 21. The mannuronic diacid saccharidecomposition according to claim 1, wherein the total weight of mannuronicdiacids wherein m+m′=2 is 30-50% of the total weight of the composition.